An equal deficit of 700 kcal∙day−1 was prescribed, restricting diet only, or moderately restricting diet and adding exercise, producing four groups; normal diet (NORM); low-carbohydrate-high-fat diet (LCHF); normal diet and exercise (NORM-EX); and low-carbohydrate-high-fat diet and exercise (LCHF-EX). Linear Mixed Models were used to assess between-group differences. The intervention resulted in an average 6.7 ± 2.5% weight-loss (p < 0.001). Post-intervention % fat was lower in NORM-EX than NORM . NORM-EX reached lower values in total cholesterol than NORM , and LCHF-EX . Post intervention triglycerides levels were lower in NORM-EX than NORM . The low-carbohydrate-high-fat diet had no superior effect on body composition, 2peak or cardiovascular risk factors compared to a normal diet, with or without exercise. In conclusion, the intervention decreased fat mass, but exercise improved body composition and caused the most favorable changes in total cholesterol and triglycerides in the NORM-EX. Exercise increased cardiorespiratory fitness, regardless of diet.We assessed the effect of weight-loss induced with a low-carbohydrate-high-fat diet with and without exercise, on body-composition, cardiorespiratory fitness and cardiovascular risk factors. A total of 57 overweight and obese women (age 40 ± 3.5 years, body mass index 31.1 ± 2.6 kg∙m The prevalence of overweight and obesity is high due to chronic positive energy balance. In 2016, over 1.9 billion people were overweight . OverweiDietary management with calorie restriction is the most applicable tool for weight-loss . Substan−1 to achieve a gradual and steady loss of 0.5–1 kg a week. Exercise without weight-loss has a profound effect on physical health in overweight and obese individuals [Exercise can be an efficient tool for weight-loss, although the exercise volumes needed for a large weight loss may not be practical or sustainable in overweight and obese individuals . Centersividuals ,25, and ividuals . Weight-ividuals ,28,29. Mividuals ,31, to rividuals . Previouividuals . Intervaividuals ,35.There is no doubt that overweight and obesity constitute a global health hazard, but there are widely differing opinions on how to reduce fat mass and improve cardiometabolic health. Primary prevention strategies include an adoption of healthy lifestyle behaviors , and stuwww.clinicaltrials.gov as NCT04100356. The study was conducted in accordance with the Declaration of Helsinki and all participants provided written informed consent before entering the study. The study was conducted from October 2013 to April 2014 at The Norwegian School of Sport Sciences, Oslo, Norway and Atlantis Medical University College.The study was a ten-week intervention where a 2 × 2 factorial design was used for a combination of diet and exercise. Participants were allocated randomly to one of four groups with a combination of normal or LCHF diet, with or without interval endurance exercise: normal-diet-only intervention group (NORM); LCHF-diet-only intervention group (LCHF); normal diet with exercise intervention group (NORM-EX); and LCHF with exercise intervention group (LCHF-EX) . The stuParticipants were recruited through newspaper advertisements and social media. In total, 199 women showed interest in participating in the study and were screened for eligibility by e-mail and telephone interview. Eligibility criteria for the study were as follows: sedentary premenopausal Caucasian women, BMI 26.9–36.1, age 33–47 and living close to, or in Oslo. Exclusion criteria were pregnancy or breast-feeding, smoking or tobacco use, previous medical history of CVD, diabetes, endocrine disorder or kidney disease, and use of lipid-lowering or diabetes medication. After initial screening by email and telephone, 60 eligible participants were included. A study flow chart of enrollment and participant flow, as recommended by the Consolidated Standards of Reporting Trials (CONSORT) is presented in www.randomizer.org). Group allocation was sent to participants by email immediately after the randomization. Neither researchers nor participants were blinded to the intervention groups.The research leader and assistant performed a simple online computer-generated randomization after baseline measurements . This was achieved in the “diet-only” groups by reducing intake, and by a combination of exercise and reduction in intake in the “diet-exercise” groups. During exercise sessions, participants joined intervals until they reached an energy expenditure of 500 kcal∙session−1. The combination of treatments gave the following calculated deficit in the diet-only groups: NORM and LCHF , and diet-exercise groups: NORM-EX and LCHF-EX .Eligible subjects were allocated to four different interventions. The calorie deficit prescribed for all groups was −4900 kcal∙weekAn introductory information meeting for the respective groups was held prior to beginning the study. A motivational speech was given to prepare the participants for the intervention and general questions were answered. Participants had coaching and follow-up from trained nutritionists (BA nutrition) twice a week, either by video meeting or phone call. Total energy expenditure (TEE) was estimated for each participant, using the Harris–Benedict equation  multiplipeak with a 3-min active rest at a comfortable recovering speed (~60% of HRpeak). Approximately halfway through the session, participants were asked to rate their perceived exertion during exercise on the Borg scale [The exercise form was interval exercise for improving cardiovascular fitness, and to result in increased energy expenditure in the exercise groups. Participants in the two exercise groups attended an indoor bicycle exercise session three times a week , supervised by a qualified instructor. The exercise consisted of a 10 min warm-up, followed by 7 × 4-min intervals at a target intensity of 82–90% of HRrg scale . Heart rrg scale . EveryboThe diet recommended by the Norwegian Health Authorities , was use−1 in the first week [−1 until participants reached a maximum 100 g∙day−1 of carbohydrates . Waist and hip measurements were performed with a medical non-stretchable measurement tape and measured to the nearest 0.1 cm. Waist circumference was measured halfway between the lower point of the rib arch and the top of the iliac crest, parallel to the floor at the end of a normal expiration. Hip measurement was done at the fullest part of the hip. Measurements were repeated three times and the mean was calculated. Weight was measured on a calibrated bioelectric impedance device  (BIA).A whole-body scan was performed, using Dual-energy X-ray absorptiometry (DXA)  and analyzed by enCORE (software version 14.10.022). The body scan was done in the fasted state in the morning, wearing underwear, without any metal objects or shoes.2peak) was performed in the afternoon, in a fed state. The testing was completed using an incremental test on an ergometer bicycle . Oxygen consumption and carbon dioxide production were measured using an automatic O2/CO2 analyzer  with a breath-by-breath average calculated in 30-s intervals throughout the protocol. A two-point gas calibration, in addition to volume calibration, was done daily prior to testing. A 5-min warm-up was performed prior to the 2peak test, which started at 50 W with an increase of 15 W every 30 s until exhaustion. Oxygen uptake was measured throughout the protocol. Capillary blood was sampled one minute after termination of the 2peak test and analyzed for lactate . An increase of less than 1 mL∙kg∙min−1 after two increments in workload, combined with a respiratory exchange ratio (RER) > 1.10 and lactate above 7 mmol∙L−1, was set as the criterion for 2peak. Peak heart rate (HRpeak) was recorded  during the 2peak test and noted as the highest heart rate.Testing of peak oxygen uptake  and coagulated for 30 min at room temperature before centrifugation . Samples were stored at 4 °C for three hours before analysis at Fürst Laboratory, Oslo, Norway .www.somebody.no, Somebody AS © 2008–2016), and total energy intake, carbohydrate, fat and protein intake were calculated. This specific tool was chosen as it is user-friendly. This was important, as participants were to use it daily. Participants were provided with an online account for the entire intervention period. A total of six nutritionists supervised the participants. The nutritionists supervised participants from different groups in order to reduce bias due to subjective diet guidance. Nutritionists had access to their clients’ accounts and registered the individual energy targets. Nutritionists assessed food registration and gave advice and suggestions for food and drink according to the respective group during the intervention. Clear dietary targets were provided with individual macronutrient targets for each participant. High levels of encouragement were given by using motivational techniques including goal setting, feedback on weight loss and dietary compliance achievements [Food was self-prepared and weighed on an electronic scale (1 g precision) during the intervention. Dietary intake was registered in an online dietary registration program (evements . Each pa−1), small (1.5 mmol∙L−1), moderate (4 mmol∙L−1) and large (8–16 mmol∙L−1) defined by the manufacturer. Participants registered the results in the dietary registry program, where nutritionists could control diet compliance.To monitor ketosis and compliance in the LCHF groups, acetoacetate was estimated in morning urine with urine sticks  from day one of the LCHF diet. The sticks change color depending on the level of ketones found in the urine. Participants compared the strip to a color chart on back of the box, with a scale reading trace (0.5 mmol∙Lhttp://www.math.yorku.ca/SCS/Online/power/). Sample size was based on the effect of exercise and weight loss on glucose tolerance [The results in this paper are from a larger study where the primary endpoint was glucose tolerance (manuscript in draft). Sample size was calculated using an online calculator  with two-sided p-values and significance level set to 5%.Main analyses of outcome variables  were performed with linear mixed models to assess the differences between groups after the intervention. The models included group, time, and group*time interaction set as fixed variables. Measurements were set nested within subject and time was included as a random slope if it improved the model, which was evaluated with a likelihood ratio test. Analyses followed the intent to treat principle, with last value measured included for dropouts. We completed pairwise comparisons within (pre-post) and between comparable groups. Results for both within and between groups are presented in the result section. Differences within groups at post-measurement were adjusted for baseline measurements. Due to multiple comparisons, all pairwise comparisons were also assessed with Bonferroni adjustments. Assumptions were examined with visual inspections of residuals and model assumptions were considered met. Descriptive analysis and differences between groups were assessed using a n = 4) during the intervention. One participant withdrew due to her work situation, two gave no reason for withdrawal and one participant did not adhere to the diet protocol and dropped out , with no differences between groups after the intervention. All four groups achieved a weight loss > 5% of the initial weight. The NORM group lost 5.2 ± 2.3 kg, whereas the other diet-only group LCHF lost 6.2 ± 2.1. The exercise groups, NORM-EX and LCHF-EX lost 5.5 ± 2.2 kg and 6.7 ± 2.3 kg, respectively. Weight loss at six weeks was similar in all groups with no differences between the groups; NORM 4.1± 1.7 kg, LCHF 5.0 ± 1.9 kg, NORM-EX 3.7 ± 1.7 kg and LCHF-EX 4.6 ± 1.6 kg.p < 0.001) and the LCHF group showing the largest loss .The intervention resulted in a loss of fat mass (FM) in all groups, but no between-group differences were seen . The meap < 0.001), where the other diet-only group, LCHF, had a large reduction of 33% (p < 0.001).In regard to visceral fat, there was a reduction in all groups in response to the intervention, where the mean reduction was 24%, but no between-group differences were observed. The NORM group had the least reduction in visceral fat, of 9.1% (p < 0.001). Between-group comparison showed a difference in % fat comparing NORM with NORM-EX (p = 0.024), where NORM-EX had a large reduction, and the NORM group had an increase after the intervention.All groups except the NORM group achieved a reduction in % fat. The LCHF group had the greatest reduction of 5.9%  and the NORM group showed a 2.3% decrease (p = 0.044). The decrease in the LCHF-EX group was 2.3% but did not reach statistical significance (p = 0.067).No difference in waist-to-hip ratio was observed when comparing groups after the intervention . The NORp < 0.001) and the NORM-EX group had a 9.6% increase (p < 0.001) in 2peak. In contrast to the exercise groups, the diet-only groups showed a 5.3% (NORM p < 0.01) and 5.4% (LCHF p < 0.001) reduction in 2peak in L∙min−1 (within-group). Between-group comparison showed a difference comparing the NORM-EX group with the NORM group (p = 0.001) after the intervention, due to an increase in the NORM-EX whilst the NORM group had a reduction in response to the intervention.Both exercise groups showed a within-group improvement in cardiorespiratory fitness in response to the intervention . The LCHp = 0.003) and the LCHF-EX group (p = 0.004) after the intervention.The intervention resulted in a reduction in total cholesterol in all groups . Betweenp = 0.005, NORM-EX p = <0.001, LCHF-EX p = 0.001, p = 0.018).Within-group comparison showed that all groups except the LCHF group had a decrease in LDL after the 10-week intervention .Within-group comparison showed that all groups had a reduction in HDL cholesterol in response to the intervention: NORM 19%, LCHF 13%, NORM-EX 21%, LCHF-EX 13%, (p = <0.001) and the NORM-EX had a 37% decrease (p = <0.001). No changes were seen in the NORM and LCHF groups. Between-group differences were seen between the NORM-EX and NORM groups after the intervention (p = 0.030).Within-group comparison showed that the two exercise groups achieved a reduction in TG . The LCHp = 0.007) increase and the LCHF group had an 8.8% increase (p = 0.004). No difference was seen between-groups , −901 kcal∙day−1 in the LCHF group (37%), −829 kcal∙day−1 in the NORM-EX group (42%) and −796 kcal∙day−1 in the LCHF-EX group (43%). No difference was seen between-groups in energy intake during the intervention. When combining restriction in diet and adding exercise expenditure, the exercise groups had a larger calculated deficit per day than comparable diet-only groups. The LCHF-EX had a total deficit of −1003 kcal∙day−1 and LCHF had −901 kcal∙day−1 (p = 0.006), whereas the NORM-EX group had a total deficit of -1029 kcal∙day−1, and the NORM group had a deficit of −893 kcal∙day−1 (p = 0.041). No differences were seen between the diet-only groups, or when diet + exercise groups were compared.Within-group change showed that all groups reduced their energy intake during the intervention . The enep <0.001). No significant change in E% carbohydrate was observed in the NORM groups. Between-group differences were seen when comparing NORM vs. LCHF (p < 0.0001) and NORM-EX vs. LCHF-EX (p < 0.0001) after the intervention.As expected, a reduction in E% carbohydrate was observed in the LCHF groups during the intervention . The avep < 0.001), and 61% in the LCHF groups (p < 0.001).Within-group changes were seen in all groups, where all groups increased their E% intake of proteins . The E% p < 0.001) and NORM-EX vs. LCHF-EX (p < 0.001).Between-group differences were seen after the intervention when comparing NORM vs. LCHF (p < 0.001) and LCHF-EX vs. NORM-EX (p < 0.001)  .p < 0.001, p < 0.001). Between-group differences were seen when comparing NORM vs. LCHF diet-only groups, with a higher E% fat in the LCHF group than in the NORM group . Changes in E% saturated fat were observed in three of the groups after the intervention (within-group); the NORM group had a 25% reduction, and the LCHF group had a 25.5% increase. No change was seen in the LCHF-EX group. Between-group differences were seen when comparing LCHF groups with their respective NORM groups. During the intervention, the LCHF group had a higher E% intake of saturated fat compared with the NORM group (p < 0.0001), and the LCHF-EX group had a higher intake than the NORM-EX group  from Day 2 in participants in the LCHF groups. Ketosis gradually declined when the amount of carbohydrate was increased each week throughout the study. Ten participants from each LCHF group reported that ketosis results in the dietary register program. The participants in the LCHF-EX group had on average 49 days out of 70 in ketosis and the LCHF group had 37 days out of 70. The number of participants in ketosis declined gradually throughout the intervention. The estimated urine ketone levels during the period spent in ketosis for the LCHF-EX and LCHF groups were 3.4 ± 2.8 mmol∙L−1 (n = 10) and 2.1 ± 1.3 mmol∙L−1 (n = 10) respectively.The carbohydrate restriction resulted in an increase in acetoacetate , mild headache (n = 12) and lack of power during bicycle sessions (n = 8) during the first two weeks.No serious, harmful or unintended effects were reported. Minor non-serious and well-known side effects of the LCHF diet were reported, such as dizziness , body composition and CVD risk factors in overweight and obese females. A desirable weight loss of >5% was achieved in all groups with the prescribed calorie deficit. Previous results have shown that a modest weight-loss as low as 5% can reduce and eliminate metabolic disorders associated with overweight and obesity . The weiy groups , and in y groups . The effThe current study confirms the effect of calorie deficit on reduction in FM (kg), as all our groups achieved a reduction in FM in response to the 10-week intervention. Although there is a functional diversity of adipose tissue, there is no doubt that total body fat is a major contributor to the metabolic challenges often seen in overweight and obesity and increases the risk of CVD. Weight loss improves metabolic health, but only when fat mass is reduced, and reduction in FM has been correlated with improved health and reduction in metabolic disorders . Body fa−1 (−1 to help offset loss in LBM [−1 was provided [−1 during the intervention (We observed a loss in LBM (kg) in all groups, which is identified as an adverse effect of weight loss . The rol−1  which iss in LBM , whereasprovided . All ourrvention . A possiBoth exercise groups, as well as the LCHF group, achieved favorable changes in % fat. The % fat did not change in the NORM group (without exercise), despite a significant weight reduction. This finding is unexpected, as previous weight-loss research has shown a reduction in primarily FM, although combined with some reduction in LBM ,58,59. T−1 was similar in the two groups. However, this supports the recommendation that participants in weight-loss programs should be encouraged to participate in regular exercise.The lack of improvement in % fat in the NORM group in response to the intervention, resulted in a between-group difference for the NORM and NORM-EX groups after the intervention. However, the reason behind this absence of reduction in % fat in the NORM group can only be speculated upon at this time, as the combination of macronutrients, calorie deficit and the amount of protein g∙kg2peak. The increase seen in the LCHF-EX group is similar to results from a study done on overweight men [2peak confirms the positive effect of exercise on cardiovascular fitness and the results are in line with improvements reported in other studies [peak (vigorous activity) during exercise sessions, with 53% of the exercise time at 80% HRpeak or above. It has been shown that interval exercise at higher intensities is superior to continuous exercise to gain improvements in 2peak [Both exercise groups improved their oxygen uptake markedly in response to the intervention and it is noteworthy that all participants in the exercise groups achieved an increase in ight men , whereasight men . The obs studies . The goaV˙O2peak . The intV˙O2peak . Our resThree of the groups achieved improvements in their LDL levels, whereas the LCHF group did not. Although weight loss is a recommended method to reduce LDL levels in overweight and obese people , the inaIn contrast, studies with overweight participants have shown improvement in the LDL profile after an LCHF diet, where the participants already had high LDL levels and a distorted lipid profile prior to the study . Both ou−1 per kg lost after entering a weight-stable condition was observed [The reduction in HDL seen in this study has been demonstrated in previous highly controlled weight-loss studies, where both non-HDL cholesterol and HDL were reduced . Our resobserved . As HDL observed , the redobserved , the incWithin-group changes showed a decrement in total cholesterol in both NORM and LCHF groups; however, the HDL levels decreased concurrently. This resulted in no significant changes in the total cholesterol/HDL ratio, and levels that were still above the optimal level (<3.0) after the intervention. An increase was seen in the total cholesterol/HDL ratio in the NORM and LCHF groups, which indicates an increment in the CVD risk . The aug−1), and further improvement can be difficult to achieve during such a short intervention. For comparison, the LCHF-EX group showed a reduction in TG in response to the intervention. This difference in responses in the LCHF groups was not significant when comparing those groups. Although within-group comparison showed the positive effect of exercise, we cannot conclude with exercise as superior to LCHF diet only when it comes to improvements in TG. However, the effect of the intervention in the LCHF-EX group is in line with results from a meta-analysis of randomized controlled trials that reported an 11% reduction in TG after aerobic training in obese and overweight participants with a modest weight loss [The exercise groups achieved a large reduction in TG in response to the intervention, while the diet-only groups did not show changes in TG levels. Weight loss has been associated with an improvement in lipid profile in overweight and obese individuals with a decrease in TG ,12, and ght loss . Furtherght loss . Althougght loss , our res−1, the LCHF group underreported 93 kcal∙day−1, the NORM-EX group underreported 368 kcal∙day−1, whereas the LCHF-EX group correctly reported kcal∙day−1, as estimated weight loss in regard to kcal reported was the same as achieved weight loss. However, these calculations have to be interpreted with care and can only give an indication of compliance to the diet. First, the prescribed calorie intake was calculated from kcal requirements estimated from BMR (the Harris–Benedict equation produced by DXA), multiplied by the PAL coefficient for each participant, based on a 24 h individual log  at the beginning of the trial may have delayed the weight loss by a couple of weeks, and therefore a longer trial may be necessary to detect differences between groups. Registration or control for any additional activity was also a limitation in our study. Further on, it is also important to consider whether the intervention arms received similar intensity and attention, as the exercise groups met three times weekly during exercise, where diet-only groups did not have a similar program and communicated mainly through social media.A 10-week weight loss program with exercise and/or LCHF diet showed improvements in different CVD risk factors in all groups, while weight loss achieved with exercise and diet produced better overall results than a normal diet only. Between-group comparison showed that the normal diet-only did not induce a loss in % fat tissue in response to the intervention. A normal diet in combination with exercise is superior to a normal diet-only when it comes to reduction in % fat tissue.The intervention showed that exercise, independent of diet, has beneficial effects on cardiorespiratory fitness, showing between-group differences between NORM-EX and NORM. A normal diet in combination with exercise caused greater improvement in total cholesterol, LDL and TG compared to the other groups, but also the largest decrease in HDL. The concurrent decrease in HDL in all groups stalled improvements in the total cholesterol/HDL ratio. As the NORM-EX group had the highest reported calorie deficit of 1029 kcal and the largest drop in HDL, one can question if the kcal deficit should be kept at a lower level to avoid HDL falling below levels for primary prevention, and emphasize participation in exercise beyond weight loss, to increase HDL levels.In conclusion, even with improvements in CVD risk factors in all intervention groups, a between-group comparison showed that exercise had beneficial effects on body composition in the NORM-EX group, in addition to causing the most favorable changes in total cholesterol and triglycerides. Nonetheless, the positive effect of exercise on CVD risk factors comparing the NORM-EX vs. NORM group was not detectable when comparing the LCHF-EX vs. LCHF group. The novelty of these findings is that the LCHF diet used in the study can blunt the aforementioned positive effect of exercise on CVD risk factors. However, even though total cholesterol and LDL were better in NORM-EX compared to LCHF-EX, both lipids reached levels below the primary prevention levels in the LCHF-EX group in response to the intervention. Therefore, the combination of LCHF diet and exercise used in this study is an adequate method to improve body composition and CVD risk factors in overweight and obese females.


With the widespread clinical application of the five-level version of the EuroQol five-dimensional questionnaire (EQ-5D-5L), whether the questionnaire scores are responsive to changes in patients’ health and how much changes in questionnaire scores represent patients’ real health changes require consideration. Consequently, we assessed responsiveness and estimated the minimal clinically important difference (MCID) of the EQ-5D-5L in surgically treated patients with cervical intraepithelial neoplasia (CIN) to determine the relationship between MCID and minimal detectable change (MDC).We conducted a longitudinal, observational study. Participants were patients with CIN from the gynecology inpatient department of a grade-A tertiary hospital in Shihezi, Xinjiang, China. Participants completed the EQ-5D-5L and the Global Rating of Change Questionnaire (GRCQ) at baseline and one month post-surgery. The Wilcoxon signed-rank test was used to compare EQ-5D-5L scores pre- and post-treatment. We calculated the effect size (ES) and the standardized response mean (SRM) to quantitatively assess responsiveness. Distribution-based, anchor-based, and instrument-defined methods were used to estimate MCID. MCID to MDC ratios at individual- and group-levels were also calculated.p < 0.05) at follow-up as compared to baseline respectively. The ES and the SRM of the index value were 0.47 and 0.42 respectively, indicating small responsiveness; while the ES and the SRM of EQ VAS were 0.50 and 0.49 respectively, indicating small to moderate responsiveness. The average (range) of MCIDs for index value and EQ VAS were 0.039 (0.023–0.064) and 5.35 (3.12–6.99) respectively. These values can only be used to determine whether patients have experienced clinically meaningful health improvements at the group level.Fifty patients with CIN completed the follow-up investigation . The index value and EQ visual analogue scale (EQ VAS) of the EQ-5D-5L improved by 0.025 and 6.92 (all The EQ-5D-5L has only small to moderate responsiveness in post-surgical patients with CIN, and the MCIDs developed in this study can be used for group-level health assessment. However, further study is needed concerning health changes at the individual level. Cervical intraepithelial neoplasia (CIN) is a general term for cervical precancerous lesions, including low- and high-grade squamous intraepithelial lesions (HSIL) . StudiesThe EQ-5D is a simple, generic, and standardized instrument for HRQoL measurement . At presIn previous studies, clinical efficacy was generally judged based on the statistical differences in PROs; however, it could not indicate whether they were clinically significant . The minLogically, MCID should be distinguished from the measurement error and therefore associated with the minimal detectable change (MDC). MDC represents the minimum change in the questionnaire scores required for real health changes and is mathematically related to the measurement error . ThroughPrevious studies have confirmed the small to moderate responsiveness of the EQ-5D-5L in patients with pulmonary embolism , deep veThe purposes of this study were (1) to evaluate the responsiveness of the EQ-5D-5L in patients with CIN who underwent surgery, (2) to estimate the MCID of the EQ-5D-5L, and (3) to analyze the relationship between MCID and MDC.This was a longitudinal, observational study. Participants were recruited from the gynecology inpatient department of a grade-A tertiary hospital in Shihezi, Xinjiang, China between November 2018 and August 2019. Inclusion criteria were (1) a positive cervical tissue biopsy result diagnosis of CIN as determined by a professional gynecologist as the primary admission diagnosis for the first time; (2) aged > 18 years; (3) Han ethnicity; (4) untreated before the baseline investigation; (5) the ability to express inner feelings clearly; (6) no severe comorbidities, mental illness, or cognitive impairment; and (7) willing to participate in this study. Exclusion criteria were (1) no CIN-related surgical treatment during hospitalization and (2) the establishment of invasive cervical cancer as the pathological diagnosis upon discharge.The baseline investigation was conducted through face-to-face interviews with patients when they were admitted, and the follow-up visit was performed one month after the surgery by telephone. The same investigator was responsible for both surveys. Investigators were postgraduates with a medical background and had been trained professionally.Age, marital status, education level, body mass index (BMI), medical insurance, and household income were obtained through face-to-face interviews with patients. The disease duration, histopathological results, and surgical approach were collected through electronic medical records.no problems, slight problems, moderate problems, severe problems, and unable to/extreme problems [The EQ-5D-5L consists of a short descriptive system and the EQ VAS. The descriptive system comprises five dimensions, each describing a different aspect of health: mobility, self-care, usual activities, pain/discomfort, and anxiety/depression. Each dimension has five response levels of severity: problems . After rproblems . The larproblems , 29. EQ The GRCQ is an external anchor for determining the MCID of questionnaire scores, which contains only one question : “How doParticipants’ characteristics were described by mean ± standard deviation (SD) and numbers and percentages (%). Comparisons of baseline and follow-up scores of the EQ-5D-5L were made using the Wilcoxon signed-rank test.Effect size (ES) and standardized response mean (SRM) were used to evaluate responsiveness, which were classified as per Cohen’s d standard : < 0.2, There is still no consensus on the best method for estimating MCID ; howeverIn the distribution-based method, 0.5SD and 1 standard error of measurement (SEM) for MCID is calculated as follows: t-retest . Based ot-retest . The anct-retest , 39.The instrument-defined method is based on the average of index value differences in the descriptive system of the EQ-5D-5L between the baseline health state and single-level transitions to other health states . MCID es95%(ind) measures the smallest detectable change of scores that are beyond the measurement error, at the individual level [95%(group), is equal to MDC95%(ind) divided by 95%(ind) and MDC95%(group) were calculated to illustrate the relationship between MCID and MDC [At the 95% confidence level, al level . Accordiple size . Ratios  and MDC . If the  and MDC .p < 0.05 was considered significant.SPSS (version 24.0) and R studio were used for statistical analyses, and A total of 110 patients were invited to participate in the study, of which 68 met the inclusion criteria and accomplished the baseline investigation. Fifty (73.53%) patients completed the follow-up visit on average of 32.28 ± 1.43 days after surgery. The reasons for non-completion were “no CIN-related surgical treatment during hospitalization” (n = 1), “discharged diagnosis of cervical invasive cancer” (n = 9), “not contacted” (n = 5), and “rejected” , had at least a junior school education (88.00%), had a BMI within the normal range (52.00%), and had medical insurance (98.00%); however, only 4.00% of patients had a moderate household income. Nearly all of the patients (94.00%) had HSIL, of which carcinoma in situ accounted for 23.40%, and cervical cone resection was the main surgical approach , 0.004 (p = 0.405), and 0.018 (p = 0.010), respectively, which indicated an improvement of these dimensions at follow-up  after treatment, respectively. The ES and the SRM of index value were 0.47 and 0.42 respectively, indicating a small responsiveness; and the ES and SRM of EQ VAS were 0.50 and 0.49 respectively, indicating small to moderate responsiveness. In patients who responded to the question on GRCQ transition rating as “improvement” (including “a little better” and “much better”), the index value change was positive ; i.e., ameliorating the HRQoL. ES and SRM were 0.59 and 0.67 respectively, suggesting a moderate effect. EQ VAS presented similar results as index value. At follow-up, EQ VAS exceeded baseline 9.27 (p = 0.001) on average, and it had moderate responsiveness . As for patients who were “about the same,” the change in index value and EQ VAS were 0.010 and 4.37 respectively; however, these were non-significant differences , and both were small even no responsiveness  and MDC95%(group). The index value and EQ VAS have MDC95%(ind) of 0.064 and 16.44, and MDC95%(group) of 0.009 and 2.32. The ratios of MCID to MDC95%(ind) of index value and EQ VAS were all < 1. This illustrated that MCID cannot discriminate the score change of the EQ-5D-5L from the measurement error at the individual level. Nevertheless, the ratios of MCID to MDC95%(group) for index value and EQ VAS exceeded 1, symbolizing that we have 95% confidence that the 50 patients in this study experienced the smallest significant improvement, and surgical treatment could be used as an effective treatment for patients with CIN at the group level. The ratios highlight the fact that the instrument-defined method and the distribution-based method have relatively good performances in index value and EQ VAS; therefore, these two methods were recommended for the MCID estimation of the EQ-5D-5L in post-surgical patients with CIN.Table This longitudinal study of patients with CIN showed that the EQ-5D-5L was responsive to change in health after surgery, and the effect size was between small and moderate. The index value and EQ VAS after treatment were on average 0.039 and 5.35, which can be considered an improvement in health from patients’ perspective. However, the MCID estimated in this study can only represent truly meaningful change of the HRQoL score at the group levels, not the individual levels.Among all dimensions, the anxiety/depression dimension was the most improved post-surgery, and the only one with a significant score change. This is similar to the results of a longitudinal HRQoL assessment of patients with CIN by Xie et al., who assessed patients one month after treatment, and found that the average improvement in mental component summary scores (MCS) measured by the SF-36 questionnaire was higher than that of the physical component summary scores  . A possiIn all patients, the positive changes in the index value and EQ VAS also coincided with other studies. A prospective study of Chinese patients with CIN conducted by Zhao et al. found that EQ-5D scores 1 month after treatment were significantly better than at baseline . TherefoTwo of the most commonly used indicators of responsiveness—ES and SRM—were used to estimate the degree of change in patients’ health –46. The Some researchers believe that responsiveness may depend on the direction of changes in health state and the individuals’ health state at baseline . The curResponsiveness of the EQ-5D-5L in patients with improved health states was also studied in other populations; however, the results were inconsistent. In patients with acute asthma who underwent one month of treatment and self-reported improved health states, the index value had moderate to large responsiveness with the effect size ranged from 0.63 to 0.95 . GolickiMCID is a vital component of the questionnaire application. Previous studies have utilized the mean change of MCID scores in the anchor-based method , 53; howBesides the distribution-based and anchor-based methods, the instrument-defined method can also be used to triangulate the MCID. Luo et al. used the instrument-defined method to estimate the MCID for the EQ-5D-3L, and the result was parallel to the published estimate; therefore, the instrument-defined method was regarded as an effective method for MCID estimation . Owing t95%(ind). The current results should be further validated in patients with the same pathological grade and the same disease duration.Concerning the relationship between MCID and MDC, the results demonstrated that the MCID estimated for index value and EQ VAS by the three methods can, at the group level, explain that the score change was a result of health changes rather than measurement error. However, MCID of index value and EQ VAS both cannot account for individual health changes at the 95% confidence level, possibly because of the inclusion of patients with different histopathological histories. In this study, the proportion of patients with carcinoma in situ was 22.00%. Although this belongs to CIN , compareThis study had several advantages. First, we used a combination of qualitative and quantitative approaches to assess responsiveness, which increases the credibility of the results. Second, in addition to the distribution-based and anchor-based methods, using the instrument-defined method for MCID estimation highlights the value of our results. Third, we analyzed whether the MCID estimated by each method can reflect true health changes at individual and group levels, which allowed us to determine the reliability of MCID and avoid the incorrect application or interpretation of the MCID. Although judging whether MCID differs from the measurement error is a logical next step after MCID estimation , only a This study also had several limitations. Apart from the GRCQ, a disease-specific questionnaire was a commonly used anchor in previous studies , 38; howThe EQ-5D-5L was responsive to surgically treated patients with CIN but with a small to moderate effect size. The results yielded an index value of 0.039 and an EQ VAS of 5.35. The analysis of the relationship between MCID and MDC revealed that the MCID developed for index value and EQ VAS can only determine whether patients actually experienced meaningful health changes at the group level; therefore, further study is needed concerning changes at the individual level.


Compared to conventional enzymatic cell detachment, hADSCs cultured on and detached from a CDP-coated cell culture dish showed similar cellular properties. We further performed in vivo experiments on a mouse hindlimb ischemia model (1.0 × 106 cells per limb). The in vivo results indicated that hADSCs retrieved from normal cell culture dishes and CDP-coated cell culture dishes showed analogous therapeutic angiogenesis. In conclusion, CDP could be applied to a pH-responsive cell detachment system as a simple and easy nonenzymatic method for stem cell culture and various cell therapies.Cell therapy usually accompanies cell detachment as an essential process in cell culture and cell collection for transplantation. However, conventional methods based on enzymatic cell detachment can cause cellular damage including cell death and senescence during the routine cell detaching step due to an inappropriate handing. The aim of the current study is to apply the pH-responsive degradation property of poly (amino ester) to the surface of a cell culture dish to provide a simple and easy alternative method for cell detachment that can substitute the conventional enzyme treatment. In this study, poly (amino ester) was modified  to show appropriate pH-responsive degradation under mild acidic conditions (0.05% ( Stem cell detachment from cell culture dishes is an essential step in the cell culture process and cell collection for stem cell therapies ,2. DespiA pH-sensitive polymer such as poly (amino ester) (PAE) is not only simple and easy to utilize without special processes, but also has eminent biocompatibility . Thus, PSodium chloride, sodium phosphate dibasic, sodium phosphate monobasic, potassium chloride, 1,4-butanediol diacrylate (BD), and 3-amino-1-propanol (AP) were obtained from Sigma  and used as received. Sodium hydroxide solutions (NaOH), hydrochloric acid solutions (HCl), dichloromethane (DCM), and ethyl ether were all purchased from Samchun . All reagents were of analytical grade and used without further purification.1H NMR spectrometer  operated at 500 MHz. DMSO (DMSO-d6) was used as solvent. The conversion between monomer A, B, and CDP was confirmed by 1H NMR. In detail, the integrated area of each peak in NMR is proportional to two parameters, which are the number of hydrogen atoms on the functional group and repetends of the corresponding functional group. Thus, NMR analysis in the mixture of products and reactants gave information about the mass ratio of the particular components composed of the specific functional group [The pH-sensitive cationic polymer CDP was synthesized using Michael polymerization as the method referred before . Brieflyal group . The Mw al group .v/v) penicillin , and 10% (v/v) fetal bovine serum . All experiments were performed on hADSCs with less than six passages.Human adipose tissue-derived stem cells (hADSCs) were purchased form Lonza  and cultured in Dulbecco’s modified Eagle’s medium  supplemented with 1% (w/v) at room temperature. After dissolving the CDP, the pH was adjusted to 7.0 with NaOH and filtered for sterilization . To coat the cell culture dishes, the CDP solution was poured into the dishes  and dried on a clean bench overnight without ultraviolet irradiation. The CDP-coated culture dishes were then washed three times with PBS  before their use in cell culture.CDP was dissolved in distilled water (pH 3.0) at 0.05%  at 37 °C for 10 min. The number of detached hADSCs was counted using a hemocytometer. hADSCs detached from NCDs using trypsin-EDTA  were used as a control.4 cells/well). After one day, cells were fixed with 4% paraformaldehyde  in PBS for 10 min at room temperature. Cell re-adhesion was also examined by phalloidin staining. For the re-adhesion test, 1.5 × 104 cells that dissociated from CDP-coated culture dishes using pH 6.0 PBS or from NCDs using trypsin-EDTA were re-seeded in 24-well culture dishes. Cells were fixed with 4% paraformaldehyde  in PBS for 10 min at room temperature. For phalloidin staining, the fixed hADSCs were stained with TRITC-phalloidin containing mounting medium , counter-stained with 4′,6-diamidino-2-phenylindole , and then examined using a confocal laser scanning microscope .Cell adhesion was examined by phalloidin staining. hADSCs were seeded on a CDP-coated or noncoated 24-well (1.5 × 103 cells/well), and cell growth rate was measured using the CCK-8 kit on days 1, 3, 5, and 7. At each time point, culture medium with 10% (v/v) CCK-8 solution was added to each well, and the plates were incubated for 3 h at 37 °C. Then, the absorbance of each well was measured at 450 nm .Cell growth rate was analyzed using cell counting kit-8 . For the cell growth rate assays, the hADSCs were seeded on CDP-coated or noncoated culture dishes , and the cells were treated with fluorescein diacetate  and ethidium bromide  24 h after seeding. The hADSCs were incubated in the FDA/EB solution for 5 min at 37 °C and then washed twice with PBS according to the manufacturer’s instructions. Dead cells were stained red owing to the nuclear permeability of EB, whereas viable cells, capable of converting the nonfluorescent FDA into fluorescein, were stained green. After staining, the samples were examined using a fluorescence microscope .The cell viability of the hADSCs cultured on the CDP-coated culture dishes was analyzed using FDA/EB staining. For this assay, the hADSCs were seeded on the CDP-coated or noncoated culture dishes . The expression of human BCL2 and BAX mRNA was evaluated to check the anti- and pro-apoptotic activity of the hADSCs, respectively, whereas the expression of human vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF2) was used to detect angiogenic factor secretion in the hADSCs. qRT-PCR was performed on a CFX connectTM real-time system  with a SsoAdvancedTM Universal SYBR® Green Supermix . The primer sequences are shown in The apoptotic activity of the hADSCs cultured on the CDP-coated culture dishes and the apoptotic activity and angiogenic growth factor secretion of the hADSCs detached from CDP with pH 6.0 PBS were evaluated by qRT-PCR. For qRT-PCR analysis, the hADSCs were seeded on the CDP-coated or noncoated culture dishes (1 × 10n = 5 mice per group). Normal cell culture dish—cultured hADSCs  and CDP—coated culture dish-cultured hADSCs  were intramuscularly injected into the gracilis muscle of the medial thigh. The physiological status of ischemic limbs was followed for 4 weeks after treatment. The no-treatment group was used as a negative control. All animal experiments were carried out in accordance with the guidelines of the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals, following protocols approved by the Institutional Animal Care and Use Committee (Sungkyunkwan University School of Medicine SKKUIACUC2020-06-11-1). All mice used in the experiments were cared for under specific-pathogen-free conditions.Hindlimb ischemia was induced in mix as previously described . Four-weA laser Doppler perfusion imager  was used for serial noninvasive physiological evaluation of neovascularization. Mice were monitored by serial scanning of surface blood flow in hindlimbs on days 0, 7, 14, 21, and 28 after treatment. Digital color-coded images were scanned and analyzed to quantify blood flow in ischemic regions from the knee joint to the toe. Mean values of perfusion were subsequently calculated.n = 4 per group). RNA was reverse-transcribed into cDNA. The expression of mouse platelet endothelial cell adhesion molecule (Pecam-1) and vascular cell adhesion molecule (Vcam) mRNA was evaluated by qRT-PCR. qRT-PCR was performed using a CFX connectTM real-time system  with SsoAdvancedTM Universal SYBR® Green Supermix . Each cycle consisted of the following times and temperatures: 95 °C for 10 s and 60 °C for 30 s. The primer sequences are shown in For qRT-PCR, total RNA was extracted from the retrieved ischemic limb tissue  in PBS and embedded in an optimal cutting temperature compound , frozen, and cut into 10 μm sections at −23 °C. After all the samples were completely sectioned, ten slides were selected out of the beginning, middle, and end part of each sample. Sections were stained with hematoxylin and eosin (H and E) and Masson’s trichrome (MT) to examine muscle degeneration and tissue inflammation.2.Sections were subjected to immunofluorescent staining by anti-CD31  and anti-smooth muscle (SM) α-actin. Fluorescein isothiocyanate-conjugated secondary antibodies  were used to visualize the signals. The sections were counterstained with DAPI and examined under a fluorescence microscope . Five pictures were randomly selected from each slide and fluorescent vessels were counted to quantify CD31 and SM α-actin positive vessels in ischemic regions. Fluorescent vessels were calculated as vessel number per mmp value of <0.05 was considered statistically significant. Results are expressed as the mean ± standard deviation for all quantitative analyses.GraphPad Prism 7 software was used for all statistical analysis in this study. Triplicate data were analyzed using one-way analysis of variance (ANOVA) with a Bonferroni test in all the experiments. Comparisons between two independent samples were performed using a two-tailed Student’s test. A The procedure for CDP synthesizing is illustrated in 1H NMR, as shown in CDP was synthesized by Michael-addition polymerization of monomer A and B. The activated double bond in monomer A was the combined amine in compound B by Michael-type step polymerization. The structure of the polymer was checked by To evaluate whether the CDP coating affects the cell adhesion, hADSCs were seeded and cultured on a dish coated with CDP, and the results were then compared with the normal cell culture dishes (NCDs). Both the optical and fluorescent images showed that the cells cultured on the CDP-coated culture dishes showed no abnormality in terms of cell adhesion compared with the cell cultured on the NCDs a.BCL-2 (anti-apoptotic) and BAX (pro-apoptotic) quantified by qRT-PCR also showed no significant difference between the two groups  and pH 6.0 PBS treated the hADSCs cultured on the CDP-coated culture dishes (PAE + pH 6.0 PBS).) Before detaching the hADSCs from the CDP-coated culture dishes, we removed the culture medium and washed the cells with pH 7.4 PBS. Then, to induce the detachment of the hADSCs from the CDP-coated culture dishes, we treated the cells with pH 6.0 PBS for 10 min at 37 °C. In optical images, we confirmed that the cells in both groups had a round shape morphology. The number of detached hADSCs was quantified by cell counting and there was no difference in both groups a.In addition, to verify the effect of pH 6.0 PBS treatment on cellular function, we detached cells from CDP-coated culture dishes using pH 6.0 PBS and then allowed them to re-attach. The cellular functions were compared with the cells re-attached after detachment from cell culture dishes using trypsin-EDTA. Two hours after re-attachment, the cells from both groups were analyzed to investigate any changes in cellular function. Gene expression from the re-attached hADSCs related to apoptosis factors  b and angIschemic hindlimb recovery facilitated by the hADSCs cultured on the CDP-coated culture dishes and dissociated with pH 6.0 PBS was evaluated using a mouse ischemic hindlimb model. We transplanted hADSCs dissociated from the cell culture dishes with trypsin-EDTA or the CDP-coated culture dishes with pH 6.0 PBS into the gracilis muscle. On day 28, the hADSCs detached from the cell culture dishes using trypsin-EDTA (NCD group) and the CDP-coated culture dishes with pH 6.0 PBS (CDP group) showed significantly improved ischemic limb salvage and limb perfusion than the no-treatment group a,b.Pecam-1 and Vcam, both related to the presence of microvessels, on the ischemic limb was quantified by qRT-PCR. mRNA expression of Pecam-1 in both the NCD and CDP groups were significantly upregulated in comparison with the no-treatment. mRNA expression of Vcam-1 in the CDP groups showed a significant increment compared with the no-treatment. However, no significant differences were found between the NCD and CDP groups in any of parameters tested. The number of microvessels in the ischemic tissues was analyzed by immunohistochemistry (IHC)  e and quary (IHC) f. The nuP groups e,f.In this study, we showed an alternative method for harvesting stem cells through enzyme-free desorption using a pH-sensitive CDP instead of the conventional trypsin-EDTA method. Trypsin-EDTA is one of the representative enzymes that can decompose cell adhesion molecules for cell detachment from the cell culture dish . TreatinWe have cultured and detached the hADSCs from CDP-coated cell culture dishes and compared the cellular effects with conventional trypsin-EDTA treatment. The CDP synthesized to coat the surface of the cell culture dish was developed from PAE and showed pH-sensitive degradation under pH 6.0 PBS. CDP was successfully synthesized with the polymerization occurring between the amine group and double bond of monomers A and B, as shown in Simple and easy cell detachment using the CDP in this study is less dependent on specific conditions such as temperature. Moreover, cellular behaviors of hADSCs cultured on CDP and detached with pH 6.0 PBS showed no differences against the cells cultured on the normal cell culture dish and detached with trypsin-EDTA. Additionally, it is relatively convenient to coat CDP on the surface of the cell culture dish compared to other polymers developed for cell detachments ,9,10. CoIn summary, we showed that we were able to retrieve the hADSCs from cell culture dishes in an enzyme-free manner through a mild pH condition by applying CDP to the cell culture system. The hADSCs cultured on CDP-coated culture dishes and detached through pH 6.0 PBS had similar cellular functions to the cells cultured on cell culture dishes and detached with trypsin-EDTA. Additionally, we also confirmed the similar in vivo therapeutic angiogenesis from both hADSCs retrieved from CDP-coated culture dishes, and normal cell culture dishes with enzyme treatment. Our CDP system may provide a new alternative stem cell harvesting platform to cell therapies.


Small-angle X-ray scattering and transmission electron microscopy showed the phase formation in the sequence of hexagonally packed cylinders (HEX) to lamellar layers (LAM) to inverse hexagonally packed cylinders (iHEX) in this series of block polymers. Wide-angle X-ray diffraction experiments proved that the fluorodomains of the LAM phases and the matrix of iHEX phases contained layered structures formed by the crystallization of fluorosegments. During heating, the self-assembled lattice remained intact even after the melting of fluorodomain, with barely changed lattice parameters. Such hierarchical structural formation was understood by chain conformation and domain interaction, which may provide new insight into the molecular design of advanced materials.Fluorocontaining polymers bearing special properties are unique and important materials in modern society. In this work, we focused on the phase behavior and phase diagram of poly(styrene- Recently, incorporating fluorous molecular nanoparticles into polymers further showed unconventional phase structures and phase behaviors. Dong et al. [block-ethylene oxide) (PS-b-PEO) block copolymer which showed the formation of concentric lamellae by truncated wedgelike building blocks. Hsu et al. [Fluorinated compounds possess unique aspects that are far different from those of their alkyl analogs, such as good chemical and thermal stability, low surface free energy, low fraction efficiency, biocompatibility, and nonsticky behavior ,2,3,4,5.60 . Consequ60 ,18,19,2060 ,24,25,2660 ,18,27 sy60  reportedg et al. ,30 reporu et al.  further block-perfluorooctylethyl acrylate) (S-b-F) block polymers bearing perfluorooctylethyl acrylate (FOA) segments on the side-chain position, which also resemble side-chain liquid-crystal-mesogen-bearing compounds [Previously, we reported hierarchical lamellar phases formed in a series of poly and melting temperatures (mT) were measured from the second heating profile to eliminate any influence from the thermal history. Small-angle X-ray scattering (SAXS) and wide-angle X-ray diffraction (WAXD) were recorded at Beamline 16B1 of the Shanghai synchrotron radiation facility (SSRF). The X-ray wavelength is 0.124 nm. Sample-to-detector distance was calibrated using silver behenate crystal. Transmission electron microscope (TEM) was detected by Tecnai F30  with an accelerating voltage of 120 kV. The samples were microtomed and stained by freshly prepared RuO4 (0.5 wt.%) vapor at room temperature for 30 min after transferring to carbon-coated copper grids.Differential scanning calorimetry (DSC) was performed on a Q100 . Glass transition temperatures (b-F block polymers. 1H NMR (proton nuclear magnetic resonance) spectrum and gel permeation chromatography (GPC) overlay of the S-b-F block polymer. The polydispersity of synthesized polymers was lower than 1.25, with unimodal GPC peaks. The chemical structure of the S-b-F block polymers and the corresponding cartoon illustrations are shown in 1H NMR spectroscopy, and the volume fractions of poly perfluorooctylethyl acrylate (PFOA) and PS were calculated using Equations (1) and (2); density measurement was according to our previous report [gT) and melting temperature (mT) of this series of S-b-F block polymers were first studied using DSC under a heating/cooling rate of 10 °C/min in N2 atmosphere. mT of 80 °C, and the PFOA segments in block copolymers showed various degrees of melting-point depression. For the sample of S74-b-F7, no mT could be identified. In this case, there was a clear gT of 95 °C, which was in excellent agreement with the PS homopolymers. It was speculated that the PFOA segments were too short for crystallization in the phase-separated domain in S74-b-F7, whereas the glass transition of polystyrene in other samples was overwhelmed by the crystal melting of the PFOA segments. Results suggest that the block copolymers may have undergone complete phase separation.Detailed synthesis and characterization can be found in our previous research . We firss report . Resultsb-F block polymers, we used the SAXS and WAXD techniques. Samples were typically prepared by thermally annealing the freeze-dried powder, first at 120–140 °C (which was higher than the gT of PS and mT of PFOA segments) for 24 h to drive the phase separation, and then at 65 °C to further crystallize the PFOA segments for 24 h to fully develop the hierarchical structures. For relatively symmetric block polymers with volume fractions of PFOA (fPFOA) ranging from 0.42 to 0.55, lamellar structures were formed with various d-spacings  phase in S74-b-F42 and S35-b-F45. To probe the detailed morphologies of the different S-spacings A. When l profile C. Meanwhχ), as shown by the high stability of the phase-separated lamellar structure in the temperature-dependent SAXS experiments [b-F42 sample with a hexagonally packed cylinders (HEX) phase. As shown in χ value existed between two blocks. Results suggested that it may be useful in the pursuit of nanostructures with ultrasmall domain sizes [−1. This meant that FOA segments were densely packed in the phase-separated morphology and could be treated as a hard substrate with high density [4 before imaging. b-F7, S74-b-F17, and S74-b-F42, respectively. In a previous study, we inferred that PS and PFOA blocks may have high Flory–Huggins interaction parameters  of the PS segments in each block copolymer. We used the radius of gyration (Rg) of the unperturbed polystyrene chain as an estimate of its size in the bulk. It could be calculated using a fitted equation on the basis of experiment data from light scattering , where d was the d-spacing of the first-order peak determined for the corresponding phases [fPS. In the HEX phase, the shape of the PS matrix was irregular, with different degrees of stretching. Since hexagonally packed cylinders are a 2D lattice, PS stretching was evaluated in two dimensions; thus, 2/3 of the power of the volume fraction was introduced to evaluate the average stretching [Polymers usually undergo thermal expansion upon heating , and theg phases . In the retching .(3)S=2dS values are summarized in The calculated A0) is also an important parameter to evaluate the chain conformation and stability of phase structures [A0 could no longer correctly reflect the stability of the phase. By contrast, the influence of the PS molecular weight was normalized in stretching ratio S. Therefore, the stretching ratio could serve as a proper indication of the conformation of PS in the assembly.The value of the interfacial area per molecule (ructures . While iS values for all S-b-F block copolymers with different fPS are plotted in S value larger than 1.0, meaning that the PS chain was stretched. This is consistent with the fact that PS was in the minority phase, and chains had to adopt an extended conformation stretching from the interface. For the LAM and HEX phases, the S values were either smaller than or not much deviated from 1.0. This means that PS chains were either compressed or relaxed. In most LAM cases, it was smaller than 1.0, which suggested that PS chains were likely compressed to a certain extent. This is probably due to the crystallization of the PFOA domain, whose extended conformation may provide more interface for the PS chains, which consequently have to compress in order to fill in space. Upon melting, the expansion of the PFOA domain in the direction perpendicular to the normal layer and the thermal expansion of the PS phase may counter each other. This may also explain the reason that d-spacing of the phase-separated nanostructure showed little change. b-F block copolymers bearing a side-chain perfluoroalkyl segment with a volume fraction varying from about 0.2 to 0.80. The phase structures were probed using the SAXS and TEM techniques, and the phase diagram covering HEX, LAM, and iHEX phases was tentatively constructed. When the PFOA block served as the minority part within a HEX structure, the crystallization of the perfluoroalkyl chain could have been compressed to some extent. However, if the PFOA molecular weight was significantly large, the long chain still crystallized. On the other hand, when the PFOA formed the matrix in an inversed HEX phase, the crystallization of the perfluoroalkyl chain formed a hierarchical lamellar structure. Similar morphologies were also formed in the LAM structures with different volume fractions. Because such a block copolymer has high thermal stability, no thermal expansion was detected during heating. We can rationalize this phenomenon by treating the PFOA block as a dense matrix, and calculating the stretching of PS chain. Results showed that, in all cases, the PS chain was slightly stretched or compressed with a S value around 1.0, meaning that the conformational penalty was largely diminished under the current circumstances. Such fluorocontaining block copolymers might serve as advanced materials in future manufacturing. In conclusion, we studied the phase behavior of a series of S-


Pediatric fractures are a common injury, and treatment often includes cast immobilization. For pediatric patients being treated in a cast, cast damage is among the most common reasons patients return to the emergency room. The figure-of-eight wrapping technique interdigitates layers of fiberglass which may create a stronger cast. The aim of this study was to assess the strength of the figure-of-eight wrapping technique in comparison to the spiral wrapping technique. A total of 10 casts were wrapped with a three-inch fiberglass using the spiral technique and 10 casts were wrapped using the figure-of-eight technique. Each cast was then subjected to a three-point bending test and loaded until failure using an Instron machine. The figure-of-eight technique had an average load to failure of 278.2 + 27.6 N/mm which was similar to the spiral technique’s load to failure of 281.2 + 25.4 N/mm (p=0.795). Prior to normalizing for thickness, the load to failure of the figure-of-eight technique was 949.8 + 109.5 N, which was significantly higher than the spiral technique of 868.2 + 65.1 N (p=0.038). The figure-of-eight casts were slightly thicker than the spiral casts . This suggests that the thickness of the fiberglass cast may improve the strength. The figure-of-eight wrapping technique had similar biomechanical characteristics to spiral wrapping techniques. Providers should wrap in whichever technique they feel most comfortable performing as there is no difference in strength of the cast. If a stronger cast is desired, then thickness of the cast can be increased. Pediatric fractures are a common injury with an annual occurrence rate of 9.47 per every 1,000 children .Healthcare providers have gravitated toward stronger immobilization to withstand the day-to-day stress a cast endures. Plaster of Paris was once the primary material of casts; however, fiberglass is now the most common material used for casting, which is largely due to increased strength -6. HistoA total of 20 polyethylene foam core cylinders were used as cast models . Each cast model was wrapped in one layer of three-inch cast padding with 50% overlap. A total of 10 casts were wrapped with a three-inch fiberglass using the spiral technique and 10 casts were wrapped using the figure-of-eight technique  provides at least 80% power to detect a difference of 15% in cast strength characteristics between the figure-of-eight and spiral wrapping techniques.The figure-of-eight wrapping technique conveys similar strength to the spiral wrapping technique. The figure-of-eight technique had an average load to failure of 278.2 + 27.6 N/mm, which was similar to the spiral technique’s load to failure of 281.2 + 25.4 N/mm (p=0.795). The stiffness of the figure-of-eight technique was 180.3 + 21.8 N/mm, which was also similar to the spiral technique’s stiffness of 186.8 + 8.5 (p=0.433) . Although this was a very small difference, it had profound influence on cast strength. Prior to controlling for thickness, the load to failure of the figure-of-eight technique was 949.8 + 109.5 N, which was significantly higher than the spiral technique of 868.2 + 65.1 N (p=0.038). This suggests that the thickness of the fiberglass cast has a considerable effect on the strength. Fiberglass cast application and maintenance are instrumental in the treatment pediatric orthopedic musculoskeletal ailments. Cast breakdown is problematic and often necessitates a trip to the clinic or emergency department for cast reapplication . A stronFiberglass has many favorable biomechanical properties including its strength, weight, and radiolucency -14. A biNew techniques have been developed that combine plaster and fiberglass (hybrid cast) in efforts to harness the best properties of both materials ,15. ThisAdditional interest has focused on techniques to improve strength of fiberglass casts ,18. One The figure-of-eight wrapping technique is popular amongst cast technicians and teaching hospitals. The figure-of-eight wrapping technique consists of interdigitating the fiberglass at approximately 45 degree angles. This method is time consuming and tedious. Historically, this technique was assumed to provide additional strength to the cast construct; however, to the best of our knowledge, there are not any biomechanical assessments comparing the figure-of-eight technique to the spiral technique.This study compared the biomechanical properties of the figure-of-eight wrapping technique to the spiral wrapping technique. Our results demonstrate that both techniques provide similar load to failure and stiffness. Interestingly, the figure-of-eight casts were slightly thicker than the spiral wrapping group (0.3 mm). This small difference in thickness attributed to approximately 9% increase in cast strength. The primary limitation of this study is that it is a biomechanical study, and its translation to clinical practice is not guaranteed.While fiberglass casts are currently the gold standard and are stronger than many other cast material, they still fail. Increasing cast thickness improves strength; however, figure-of-eight and spiral wrapping techniques convey similar biomechanical properties. Further research is needed to improve cast strength and prevent early cast breakdown.


We found that the slow oscillation (SO) < 1 Hz frequency was significantly faster in both the mPFC and hippocampus in A30P mice, and Up-state-associated fast oscillations at beta (20 – 30 Hz) and gamma (30 – 80 Hz) frequencies were delayed relative to the onset of the Up-state. Spindle (8 – 15 Hz) activity in the mPFC was also altered in A30P mice, as spindles were shorter in duration and had reduced density compared to WT. These changes demonstrate that dysregulation of sleep-related oscillations occurs in young A30P mice long before the onset of cognitive dysfunction. Our data suggest that, as seen in patients, changes in sleep-related oscillations are an early consequence of abnormal α-syn aggregation in A30P mice.Changes in sleep behavior and sleep-related cortical activity have been reported in conditions associated with abnormal alpha-synuclein (α-syn) expression, in particular Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Notably, changes can occur in patients years before the onset of cognitive decline. Sleep-related network oscillations play a key role in memory function, but how abnormal α-syn impacts the generation of such activity is currently unclear. To determine whether early changes in sleep-related network activity could also be observed, prior to any previously reported cognitive dysfunction, we used mice that over-express human mutant α-syn (A30P). Recordings  Dementia with Lewy bodies (DLB) is the second most common cause of dementia after Alzheimer’s disease (AD) . ClinicaSleep-related network oscillations can be recorded using the electroencephalogram (EEG) in both humans and rodents during awake and sleep states. Sleep consists of two distinct patterns of cortical activity, REM sleep and non-REM (NREM) sleep that can be readily distinguished in local field potential (LFP) recordings. REM sleep is defined by low amplitude, oscillatory activity predominantly in the theta (4 – 8 Hz) band. In contrast, NREM sleep is characterized by a SO < 1 Hz and sleep spindles at 8 – 15 Hz . Both REIn rodents the SO can be recorded during both natural sleep , and undIn addition, spindle activity between 8 and 15 Hz also occurs on the Up-state . ChangesTo-date the majority of EEG studies in patients with DLB have been conducted during the awake state where a well described ‘slowing’ of the dominant frequency in the EEG is observed . Using mRecordings were made under urethane anesthesia, a widely used model of NREM sleep in rodents . We asseA30P mice  expressing human mutant A30P α-syn under the control of the Thy-1 promoter  were useIn Vivo Experiments) guidelines. Mice were kept in an enriched environment (cage toys) under a 12 h light:dark cycle (lights on 7 am–7 pm) with access to food and water ad libitum. Mice were group housed and experiments were commenced ∼2 h into the light (sleep) phase of the circadian cycle.All procedures described below were in accordance with the UK Animals (Scientific Procedures) Act 1986 and the European Union Directive 2010/63/EU. WT and A30P mice were housed at Newcastle University’s animal facility in a temperature- and humidity-controlled environment consistent with the ARRIVE . Mice were then transferred to a stereotaxic frame  placed on a heating pad with feedback temperature control via a rectal probe  with core temperature maintained at 36.8°C. The mice breathed spontaneously but to maintain an oxygen saturation of >95%, medical oxygen  was supplied through a tube mounted to the nose bar of the stereotaxic frame. For the duration of the surgery (20–30 min), 1% isoflurane was also provided through the mask. The isoflurane concentration was then gradually reduced and stopped when a deep plane of urethane anesthesia was achieved. The level of anesthesia was confirmed by absence of the pedal withdrawal reflex. The reflex was checked regularly and additional doses of urethane (3.3 g/kg) were given i.p. if indicated.A skin incision was made in the scalp and infused with lidocaine, before the periosteum was retracted to expose bregma. A craniotomy was drilled above the left mPFC and/or the right hippocampus. Electrodes (see below) were implanted into the left mPFC  and/or right hippocampus passing through the CA1 region of the hippocampus . The electrode was lowered ventrally through the dura using a one-axis oil-filled hydraulic micromanipulator .Multi-channel recordings were made in all subregions of the mPFC in the left hemispheres and/or the contralateral hippocampus passing through the CA1 . Single After the experiment the mouse was sacrificed by injection with Euthatal (0.2 ml i.p.). The brain was removed from the skull and post-fixed in 4% paraformaldehyde (PFA) and 0.1 M phosphate buffer solution (PBS) at 4°C, for approximately 48 h. The brain was then transferred for cryoprotection in a 30% sucrose solution for 48–72 h. Coronal sections (60 μm) were cut on a cooled vibratome  and collected in 0.1 M PBS. DAPI  staining was used to verify the position of single channel tungsten electrodes, and DiI  was used to verify the position of the silicon probes . After sThe raw LFP was band-stop filtered at 50 Hz. The LFP recordings used were concatenated sections from 30 min-long recordings taken once SO activity was clearly established. The resulting LFP recordings of SO were approximately 20 min-long. All data analysis was performed offline using custom MATLAB  scripts.t) = 0) (Up- and Down-state (UDS) detection  was perft) = 0) . To qualTime points of Up- and Down-state transitions were calculated from the UDS detection logical vector. A cycle always consisted of a Down-state and an Up-state and contained three transitions: transition 1: Up-to-Down transition  2: Down-to-Up transition  3: Up-to-Down transition . A phase vector was calculated where 0 was the time point of transition 1, 50 was the time point of transition 2, and 100 was the time point of transition 3. The time points in between were filled with linearly spaced intermediate values, so that a phase vector was achieved with linear phase progression during the Down-state, and linear phase progression during the Up-state. All UDS were then divided into 40 bins per state .z-scores  and normalized to the baseline. The instantaneous amplitudes of the analytical signal and of the Hilbert transform of the ripple band-passed signal were calculated. The instantaneous amplitude was smoothed with a 15 ms kernel. The points of the smoothed signal that crossed the upper threshold  were identified as potential ripples. Ripples with peaks less than 100 ms apart were considered to be one ripple event. The duration of the ripples was determined by the crossing points of the lower threshold . Finally, ripples that were shorter than 20 ms and longer than 400 ms were rejected.For sharp-wave ripple (SPW-R) detection we also adapted the methods described by For spindles and SPW-Rs we measured the following; (1) the average amplitude of the event from zero to the peak of the LFP normalized to the baseline. (2) Duration of spindles/SPW-Rs determined as the time between the two points that crossed the lower threshold of the smoothed signal. (3) The variability over time (CV = SD/mean) of amplitude and duration. Other variables of interest were the spindle/SPW-R latency in the normalized SO cycle, the relative spindle/SPW-R density (number of events/Up-state), and the percentage of high-amplitude spindles (amplitude > 5 × mean event amplitude for each recording). Finally, the instantaneous frequency of each spindle/SPW-R event was calculated as the number of cycles of the spindle or SPW-R band-passed LFP that crossed the upper threshold of the smoothed signal, divided by time interval between the two crossing points.pxx(f) and pyy(f) and the cross power spectral density, Pxy(f), of x and y where x is the LFP and y the spike train of the neuron.Single neurons in the mPFC were first sorted online using the Plexon Sort Client and then offline using the Plexon Offline sorter. During offline sorting, all waveforms were aligned to the global minimum and short interspike intervals (ISIs) of (<1 ms) were automatically removed. Only neurons which could be well distinguished were included in the analysis. In most cases one neuron per channel was recorded, although occasionally two neurons could be isolated. No neurons were obtained from the hippocampus. The neuronal firing time point data were down-sampled to 1 kHz. Measures of coherence between the SO band-passed LFP and the neuronal firing were obtained using the mscohere function in MATLAB. The threshold firing rate for including a neuron for analysis was set at 10 spikes/min (0.17 Hz), while for coherence analysis this was set at 30 spikes/min (0.5 Hz). The magnitude-squared coherence was calculated as a function of the power spectral densities, The time points of neuronal firing were compared to the normalized Up-Down cycle to identify whether they occurred during an Up- or a Down-state. The percentage of firing on the Up- and Down-states was calculated as a percentage of the total firing. The neuronal firing frequency on the Up-state was calculated as a fraction of the total number of spikes occurring on the Up-states divided by the length of all the Up-states combined. The neuronal firing frequency on the Down-state was calculated in a similar manner. The neuronal firing frequency for the duration of the recording was determined by dividing the total number of spikes recorded, irrespective of state, by the duration of the recording. We also calculated the ISI and coefficient of variation (CV) of neuronal firing on the Up-state for the duration of the recording. Finally, we found the maximum coherence between the SO and neuronal firing on the Up-state and identified the frequency at which it occurred at (0.1 – 0.9 Hz).U-test with a Welch exact-correction analysis was performed for the SPW-R data where normal distribution could not be achieved. Parametric data is presented as mean ± standard error of the mean (SEM), while non-parametric data is presented as median ± interquartile range (IQR).Firstly, we ensured a Gaussian distribution of data and homogeneity of variance using the Kolmogorov–Smirnov and Levene’s Test respectively. If normality could not be confirmed, we performed a logarithmic or a square root transformation of the data. Specifically, we performed a logarithmic transformation for power on the Up- and Down-states and the mean peak power for all frequency bands in the mPFC and CA1. Mixed repeated measures ANOVA (RM ANOVA) analyses was conducted where mPFC subregion (four levels) was set as the within-subjects variable and the animal genotype as the between-subjects variable. A detailed comparison of the differences between mPFC subregions was out of the scope of the current study, however we proceeded with mPFC subregions as a repeated measure to identify any mixed effects/interactions with the genotype . The outPrevious studies recording cortical activity in mice and rats using urethane anesthesia have reported sleep-state alternations between the SO (∼ 1 Hz) and a REM-like activity in which theta frequency activity predominates . We alsoF = 9.64, p < 0.01], in all subregions of the mPFC  = 5.93, p < 0.05], while the Down-state was significantly shorter in all mPFC regions  in A30P mice  = 6.36, p < 0.05] and gamma  power in the PrL and IL regions. The normalized SO cycle consists of the Down-state followed by the Up-state, where 0% in the normalized SO cycle is the beginning of the Down-state, 50% is the Down-to-Up-transition and 100% is the Up-state termination . In the PrL region the beta power peak latency was 61.5% ± 0.8% in A30P animals compared to 58.5 ± 0.84% in WT animals. In the IL subregion the beta power peak latency was 63.2 ± 0.94% in A30P compared to 58.9 ± 1.0% in WT animals. We did not find any significant differences in the high-gamma frequency band between WT and A30P mice  = 14.21, p < 0.01], in all mPFC subregions  = 22.39, p < 0.01] in all mPFC subregions  = 6.17, p < 0.05] in all subregions of the mPFC  = 4.71, p < 0.05] in the ACC and PrL regions, with a similar trend in the other subregions  = 17.57, p < 0.01]. Also consistent with the mPFC findings, the increased SO frequency in A30P animals was due to a shorter SO cycle length  composed of both shorter Up-  and Down-states   = 5.48, p < 0.05, Univariate ANOVA], but there was no change in the latency to peak beta or high-gamma power  = 4.89, p < 0.05]. Consistent with this the percentage of the total firing occurring on the Down-state  = 4.75, p < 0.05]. The neuronal firing/SO coherence  = 4.95, p < 0.05]. Our findings indicate increased in neuronal firing during the predominantly “silent” Down-state, suggesting aberrant neuronal activity.As the changes in SO and Up-state associated oscillatory activity in the mPFC outlined above could reflect changes in neuronal firing in A30P mice we compared neuronal properties in WT and A30P mice . Neuronswn-state  in A30P oherence  was alsoWe have identified a number of significant changes in the SO in both the mPFC and CA1 of the hippocampus of A30P mice under urethane anesthesia as a consequence of the expression of human mutant α-syn. The SO frequency was faster in A30P mice in both the mPFC and hippocampus as a result of the shorter Up- and Down-states. In addition, the distribution of power on the Up-state was altered in A30P mice, with an increased latency to peak power in the beta and gamma frequency bands in the mPFC and gamma band in hippocampus. We also found that in A30P mice a greater percentage of the total firing occurred on the Down-state, and there was a lower neuronal firing/SO coherence compared to WT mice. There were no changes in the total power of beta, gamma or high-gamma activity on the Up-state between A30P and WT animals in either the mPFC or hippocampus, and no change in hippocampal ripples. However, interestingly, A30P mice had impaired spindle activity, including reduced spindle duration, less high-amplitude spindles and lower relative spindle density. These early changes in sleep-related oscillations in A30P mice are occurring before any reported cognitive or motor symptoms which only emerge from ∼12 months of age , althougA key finding of the current study was an increase in SO frequency recorded under urethane anesthesia in both mPFC and hippocampus in A30P animals. Despite the known functional and anatomical differences between the mPFC subregions , the incA receptor-mediated neurotransmission (In contrast to our findings in A30P mice, studies in both the rTg4510 model of tauopathy , and thesmission . The reasmission . Howeversmission . It woulsmission . Therefosmission .2+ overload (Interestingly, in recent studies cortical optogenetic stimulation at normal SO frequencies in a mouse model of AD restored SO power and slowed down amyloid-β pathology , while aoverload . Thus it+ conductances and excitatory and inhibitory inputs (A receptor function can shorten Up-state duration (A complex interplay of different mechanisms regulates Up- and Down-state transitions including, Ky inputs  and parvy inputs . Recently inputs . Therefoy inputs . Reducedduration  so abnorduration . FurtherAs shown previously , we founIn this study, spindles predominantly occurred at the start of the Up-state as shown previously , and theSpindle impairments have been observed in DLB, PDD and PD patients . A polysThe increase in SO frequency we have seen in A30P mice could have a negative impact on the important functions of sleep. The Up-state provides a temporal framework for neurons in the hippocampus and cortex to communicate with each other and bind into the functional assemblies that underlie memory consolidation . This hiThe animals used in this study represent a prodromal model of DLB, where no measurable cognitive dysfunction has yet been reported, so we would not expect widespread and dramatic changes in sleep-related oscillations as this stage in A30P mice. Thus, for example we did not find any changes in hippocampal ripple oscillations, similar to a recent study in a murine model of AD , althougAbnormal slow wave sleep activity is thought to prevent the clearance of pathological forms of amyloid-β and tau, and even promote excess production of these peptides . If the in vitro in A30P mice (The early changes seen in our study are important for understanding the impact of abnormal α-syn pathology on network oscillations. Future work is needed to confirm that the findings reported here are also observed in A30P mice during natural sleep. In addition, it would be important to assess how changes in sleep-related activity alter with increasing age and disease progression in A30P mice as we have previously shown an age-dependent deficit in gamma frequency activity 30P mice . It woulHyperexcitability and epilepsy are well described in AD patients , 2017 anThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the UK Home Office PIL.MS: investigation and visualization. MS and BZ: formal analysis. MS, BZ, AT, J-PT, and FL: editing and revising the manuscript and reviewing and editing. J-PT, AT, and FL: supervision and funding acquisition. MS and FL: conceptualization. FL: writing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A key strategy for the primary prevention of cardiovascular disease (CVD) is the use of risk prediction algorithms. We aimed to investigate the predictive ability of SCORE (Systematic COronary Risk Estimation) and PCE (Pooled Cohort Equations) systems for atherosclerotic CVD (ASCVD) risk in Portugal, a low CVD risk country, at the 10-year landmark and at a longer, 15-year follow-up.The SCORE and PCE 10-year risk estimates were calculated for 455 and 448 patients, respectively. Discrimination was assessed by Harrell’s C-statistic. Calibration was analyzed by standardized incidence ratios (SIR).During the 10-year follow-up, 7 fatal ASCVD events (the SCORE outcome) and 32 any ASCVD events (the PCE outcome) occurred. The SCORE system showed good discrimination (C-statistic 0.83), while the PCE showed poor discrimination (C-statistic 0.62). Calibration was similar for both systems, according to SIR: SCORE, 0.3 (95% CI 0.1–0.7); PCE, 0.5 (95% CI 0.4–0.7). Globally, both 10-year fatal ASCVD risk and any ASCVD risk were overestimated in the overall population and men. However, the risk was underestimated by both systems in women. Despite an overestimation of 15-year fatal ASCVD by SCORE, the 15-year any ASCVD observed incidence was 1.8 times the 10-year incidence among men and 1.4 times among women. This acceleration of CVD risk was more relevant in the lowest classes of ASCVD risk.In this prospective, contemporary, Portuguese cohort, the SCORE had better discriminatory power and similar calibration compared to PCE. However, both risk scores underestimated 10-year ASCVD risk in women. Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality in developed countries, despite consistent improvement in outcomes . ModifiaIn 2003, the European Society of Cardiology (ESC) jointly with the European Atherosclerosis Society (EAS) published the SCORE (Systematic COronary Risk Estimation): a 10-year risk estimation system for ACSVD death . The SCOIn parallel, the American College of Cardiology (ACC) and American Heart Association (AHA) developed the Pooled Cohort Equations (PCE) that estimate a composite endpoint of 10-year ASCVD risk, instead of the SCORE-estimated 10-year fatal ASCVD risk . HoweverBesides the different prediction models available to estimate the ASCVD risk, the decision thresholds recommended for drug therapy are different for, respectively, fatal ASCVD (SCORE) and any ASCVD (PCE) . Such coIn this study, we aimed to determine the 10- and 15-year incidence of ASCVD events in a primary prevention cohort of a Southern European country. In addition, we aimed to compare the calibration and discrimination of both SCORE and PCE systems in this cohort at the predefined 10-year landmark and at a longer, 15-year follow-up, in order to estimate their external validity for clinical practice.Centro Hospitalar e Universitário de Coimbra’s Lipidology Clinic from 1994 to 2007. All patients were followed until December 2017. Among recruited participants, we selected those aged 40–79 years and with no previous history of heart disease and stroke at the screening visit. Patients with familial hypercholesterolemia or with missing information at the baseline examination were excluded, resulting in 455 individuals available for this study.We conducted a prospective single-center study, including 663 patients consecutively referred by Primary Care physicians to − 1 or the use of hypoglycemic drugs. Smoking status was dichotomized as current versus past/never smokers. On the first appointment, several baseline laboratory variables and a complete lipid profile on fasting blood samples were collected.Baseline, demographic and clinical variables were collected, including age at referral, sex, hypertension, diabetes and smoking status. Hypertension was defined as systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg, or current antihypertensive treatment. Diabetes was defined as fasting glucose ≥126 mg.dL− 1 and blood pressure ≥ 180/110 mmHg [The predictors used to estimate risk for a first fatal or any ASCVD event included age, sex, smoking status, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), systolic blood pressure, diabetes, and antihypertensive treatment. SCORE and PCE systems vary in the age ranges to which they apply. SCORE is recommended for use in the 40- to 65- year age range . In contIn SCORE, the predicted outcome is fatal ASCVD, comprising the occurrence of death from coronary artery disease, stroke, hypertension, heart failure, peripheral artery disease or aortic disease . The endn = 252) were followed for 15 years regarding fatal ASCVD and 48.9% (n = 219) regarding any ASCVD.A total of 455 patients had complete information on the components of SCORE and 448 had complete information on the components of PCE. The median (interquartile range (IQR)) follow-up time was 15 (11–17) years. All patients were followed up for at least 10 years and 55.4% (Continuous variables were expressed as mean ± standard deviation (SD). Median and IQR were used if the distribution was not normal, assessed by the use of the Kolmogorov-Smirnov test. The Student’s t-test for normal variables and the Mann-Whitney test for non-normal variables were used for comparisons among groups . Categorical variables were presented as percentages and were compared using chi-square or Fisher’s exact test.The predictive ability of the SCORE and PCE systems for the Southern European population was evaluated based on discrimination and calibration of the models. The discriminative power was compared with Harrell’s C-statistics, which takes into account the timing of events. A calibration analysis was conducted by using standardized incidence ratios (SIR), comparing the 10-year predicted event rate to the observed rate at 10-year follow-up. As all patients completed the 10-year follow-up, 10-year observed event rate was the actual rate. Regarding the calibration analysis at the 15-year follow-up, the Kaplan–Meier method was utilized to estimate the 15-year observed risk for each group. Both rates were plotted against the predicted risk, estimated as mean 10-year risk score in each group . Particin = 448). The discrimination was evaluated by Harrell’s C-statistic for each risk system-specific outcome. The pairwise differences among the C-statistics corresponding to the risk systems were calculated, and, to compensate for multiple testing, bootstrapping was used to obtain 99% confidence intervals (CI) [Concerning head-to-head comparison, an intersection of the two risk systems specific subgroups was created  .All tests were conducted using STATA 13.0 .The mean age ± SD of the study sample of 455 individuals was 57.8 ± 9.6 years and 61.8% were male. The overall baseline characteristics were similar between genders, except that women had significantly higher levels of low-density lipoprotein cholesterol (LDL-C) and a larger proportion of statin therapy  according to SCORE and the mean ASCVD risk was 13.5% (16.2% in men and 9.0% in women) according to PCE, globally indicating a population with moderate ASCVD risk  and PCE (any ASCVD) and those who did not, after calculating the 10-year risk for each patient. During the 10-year follow-up, 7 (1.6%) SCORE-specific events and 32 (7.0%) PCE-specific events occurred  ASCVD deaths and 25 (11.3%) any ASCVD events were observed. The C-statistic were 0.80 for the SCORE-specific outcomes and 0.63 for PCE-specific outcomes  Fig. .Globally, the PCE predicted 47% more any ASCVD events Fig. . ActuallIn our cohort of Portuguese patients, the evaluation of PCE and SCORE risk prediction algorithms revealed that both performed at an acceptable level. The SCORE system performed better than the PCE system in discriminating their respective endpoints. Although calibration was similar, both risk systems markedly overestimated the risk.While both risk systems address the same practical question, which individuals benefit from interventive measures, its equations differ significantly. The SCORE includes as predictors age, sex, smoking, systolic blood pressure, and TC; it is traditionally applicable in individuals from 40 to 65 years, and the predicted outcome is 10-year fatal ASCVD, disregarding non-fatal events .Existing studies regarding the SCORE risk prediction system generally reported good discrimination, consistent with our findings , 9, 24. The external validation studies that evaluate the PCE system have generated controversy around the predictive accuracy of these equations in contemporary cohorts . The PCEAccurate calibration of the risk prediction models is crucial for the success of preventive measures. Although both models overestimated the risk, when observed risks were taken into account, the accuracy of risk prediction in women was less impressive. Actually, the risk was underestimated by the PCE risk system in the low-risk (< 5%) and intermediate-risk (7.5–20%) strata. Underestimation of ASCVD risk in the female gender has only been described once previously in a Korean cohort .n = 7), especially in men (n = 2). This low ASCVDmortality rate is consistent with what has been found in a Spanish cohort of 608 non-diabetic patients on primary prevention with a similar moderate 10-year risk of fatal ASCVD (2.1%), where only 9 ASCVD deaths were reported (1.5%) [Concerning the SCORE system, the interaction between sex and events should be analyzed as an exploratory and hypothesis-generating finding, due to the very low number of 10-year fatal ASCVD events n = , especiad (1.5%) . Howeverd (1.5%) .The causes of the unexpected findings regarding the underprediction of fatal or any ASCVD risk in women and the very low number of ASCVD deaths in men are not clear. Although women from our cohort had higher levels of LDL-C, they were also treated more commonly with statins. In the past, numerous studies have shown excess mortality in women after myocardial infarction . In addiLastly, the SCORE overestimated the 15-year risk in the overall population, probably reflecting the decline of CVD mortality in the last decades . ConversIt is essential to evaluate the applicability of risk models to each population, as risk scores may perform worse in a different setting from the one they were originally derived . It is wWe acknowledge several limitations of our study. We have a limited sample size, representative of the Center region of Portugal, one of the regions with the lowest rate of CVD events in our country . This maIn this prospective, contemporary, Portuguese cohort, SCORE had better discriminatory power compared with PCE. Overall, both systems overestimated ASCVD risk at the 10-year landmark. However, among women, ASCVD risk was underestimated with both risk systems. Lastly, we also observed an acceleration of the cardiovascular risk at 15-year follow-up, particularly at the lowest risk classes. These findings may have the potential to improve risk stratification and, ultimately, treatment allocation in a primary prevention setting.